Mixtures of PRR Ligands Partly Mimic the Immunomodulatory Response of γ i Staphylococcus aureus, Enhancing Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Abstract

Recent evidence indicates the potential of gamma-irradiated (γi) Staphylococcus aureus to be used as an osteo-immunomodulator for bone regeneration. This study aims at characterizing the inflammatory milieu caused by the stimulation of γi S. aureus in immune cells and investigates its effects on MSC osteogenic differentiation. Furthermore, we aimed to recreate the immune-modulatory response exhibited by γi S. aureus by using a mixture of various synthetic pathogen recognition receptor (PRR) ligands consisting of TLR2, TLR8, TLR9, and NOD2 agonists. Human peripheral blood mononuclear cells (hPBMCs), isolated from healthy human donors, were exposed to γi S. aureus or seven different ligand mixtures. After 24 h, the conditioned medium (CM) from the hPBMCs was collected and its effects on hMSC osteogenic differentiation were investigated by assessing alkaline phosphatase (ALP) activity and matrix mineralization. The hPBMCs and their CM were also analyzed by bulk RNA sequencing and for cytokine secretion. CM from the γi S. aureus and the mixture consisting of Pam3CSK4, C-class CpG oligodeoxynucleotide (CpG ODN C), and murabutide targeting TLR2, TLR9, and NOD2 showed a fivefold increase in ALP and matrix mineralization in a donor-dependent manner. These effects were due to the upregulation of inflammatory signaling pathways, which led to an increase in cytokines and chemokines TNF, interleukin (IL)-6, IFN-γ, IL-1α, CXCL10, CCL18, CCL17, CXCL1, and CCL5. Upregulation of genes like BMP2R, BMP6R, BGLAP, and others contributed to the upregulation of osteogenic pathways in the hPBMCs stimulated with γi S. aureus and the aforementioned mix. Thus, formulations with mixtures of PRR ligands may serve as immune-modulatory osteogenesis-enhancing agents.

Keywords: PRR ligands; Staphylococcus aureus; bone regeneration; immune modulation; inflammation; pathogen recognition molecular patterns.

Figure 1

Effect of γi Staphylococcus aureus and mixtures on pathogen recognition receptor expression at the gene level in pooled hPBMCs (n = 9). (a) Illustration showing the PRRs involved in Staphylococcus aureus recognition based on literature [28]. Colored boxes correspond to colors in Table 1. (b) Heatmap showing the log2 normalized and scaled of the absolute gene count between groups. Each box depicts the technical replicates of the conditions. (c) Absolute gene count of the PRRs for all groups. Data are shown as box and whisker plots. The colored dots indicate the technical replicates used per condition.

Figure 2

Indirect effect of γi Staphylococcus aureus and mixtures on the hMSC osteogenic differentiation. (a) Study design setup. γi Staphylococcus aureus and mixtures were added to hPBMCs (n = 9) for 24 h. The conditioned medium obtained from the stimulation was pooled and used to stimulate hMSCs (n = 6) into osteogenic differentiation. The CM was added in the ratio of 1:4 (CM: ODM). (b) Alkaline phosphatase activity was measured at days 3, 7, 10, and 14 and normalized to DNA content for the controls containing CM obtained from unstimulated hPBMCs. (c) The fold change of the normalized ALP activity to the controls is shown for γi Staphylococcus aureus, (d) Mix 1, (e) Mix 4, and (f) Mix 5. The data are presented as the mean of technical duplicates performed per donor for six individual donors. Significance was tested using repeated-measures ANOVA with Sidak's post hoc test for multiple comparisons. (g) Tabular format showing the upregulation of six hMSC donors (in colored cells) for ALP activity between groups. (h) Alizarin Red S staining was performed after hMSCs (n = 5) were stimulated with CM obtained from γi Staphylococcus aureus and mixtures Mix 1, Mix 4, and Mix 5 stimulated hPBMCs (n = 9). The stained images (circular wells) show the calcium deposition in red for all groups (representative for five MSC donors). The total amount of calcium deposited per well was quantified and normalized to the control. Results show the mean + standard deviation for five MSC donors. Significance was tested using one-way ANOVA with Sidak's post hoc test for multiple comparisons. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, and ⁣^∗∗∗p < 0.001.

Figure 3

Differential gene expression analysis. (a) Illustration of the assay setup. hPBMCs (n = 9) were stimulated with either γi Staphylococcus aureus or mixtures for 24 h. RNA was isolated and processed for bulk RNA sequencing. RNA pooled from unstimulated hPBMCs (n = 9) served as the control. (b) A heatmap is showing gene expression of all groups of the top 100 most differentially regulated genes between control and γi Staphylococcus aureus. Color scale indicates normalized transformed gene expression, scaled per gene to represent deviation from the average. The colored boxes highlight the patterns observed in the genes among all the groups.

Figure 4

The gene set enrichment analysis (GSEA), comparing Gene Ontology term enrichment for γi Staphylococcus aureus and mixtures (Mix 1, Mix 4, and Mix 5) stimulated hPBMCs compared to unstimulated control. (a) Bubble plot showing the activation of inflammatory signaling pathways in all groups as compared to the control. (b) Bubble plot showing the activation of bone development and bone remodeling pathways in all conditions as compared to the control. Mix 1 was the only mixture to show an upregulation of bone related pathways. The number of the genes differentially expressed in a pathway is displayed using the size of the circle, GeneRatio displays the differentially expressed genes as a proportion of all genes in the pathway. The significance is shown as a color gradient (scale bar).

Figure 5

Characterization of proteins in CM. (a) hPBMCs (n = 9) were stimulated with either γi Staphylococcus aureus or mixtures for 24 h. The conditioned medium obtained from the stimulation was pooled and used to characterize its composition using a Luminex assay. (b) Heatmap showing the Log2 fold change from control of different proteins. All the groups were normalized to the control and transformed using Log2. Samples that were above or below the detection limit were mentioned as out of range (OOR (above range OOR > and below range OOR <)).